Lecture 2 - Examples of PMS Flashcards
What is the ratio of parental products in GC in an octad?
6:2
What are the two ratios in PMS?
5:3 1 pair not same
4:4 2 pairs not the same
(secreted colonies in yeast tetrads)
How do 4:4 aberrant ratios form?
through PMS
heteroduplexes formed in meiosis not repaired
visibilisation only visable after mitotsis
How to 3:5 ratios form?
PMS
repair to one heteroduplex, mismatch repaired
What are the four experiments by Szostak that show that original HJ required revision, because of DSBrepair?
1) dsbreaks dramatically increase recombination frequency
2) large gaps allow efficient integration
3) gap repair by recombination with intact homologous sequence
4) deletion to circular plasmid not repaired
How was it shown experimentally that dsBreaks dramatically increase recombination frequency? (szostak)
1) circular plasmid inserted into yeast chromosome get few recombinants
2) when linearized plasmid inserted get many more recombinants
How was it shown that large region in gaps of homology still allowed efficient integration?
1) showed 40 bp region of homology allowed integration of linearised plasmid
How was it shown that gaps could be repaired by recombination with homolgous sequencE?
1) insertion of linearised plasmid with 40bp homologous regions and autonomously replicating sequence
Two results:
1) led to crossover and integration with chromosome
2) no crossover gap repaired from genetic information on the chromosome
How was shown that deletion in circular plasmid not repaired?
circular yeast plasmid with region of homolgy with deletion
2)when integrated into the host chromosome regions of homolgy deletion not repaired
What gene does dsb repair require and what happens in an individual defectivE?
RAD52
individuals defective in meiotic recombination
What is the BSCR model of homolgous recombination?
1) recombination is initated by dsbreaks
2) involves double holliday junction
What is the process dhj formed?
1) Dsbs are created by Spo11 (only in meiosis)
2) Resection by 5’ to 3’ exonuclease + helicases producing 3’ overhangs
3) binding of Rad51 and Dmc1 leads to strand invasion
4) Synthesis through break and branch migration
5) second end capture and synthesis
6) ligation leading to dHJ
By what enzyme are JH cut by?
HJ resolvases to CO nCO products
In resolution of HJ how are CO products formed?
draw
In resolution of HJ how are nonCO products formed?
draw
If heteroduplexes repaired before dHJ formed how effect gene conversion?
2:6
In heteroduplex repair where is it most liely to occur?
on the chromatid where DSB occured because mismatch repair works on a strand containg a free end
What are the main types of enzymes involved in crossing over?
endonuclease to make dsb, 5’ to 3’ exnuclease, helicases, recombinase, polymerase, ligase, resolvase
Why bother with gene conversion?
Meiosis = generates genetic variation e.g. results gene conversion without crossing over can only be replicated by double cross over rare if loci are closely linked Mitosis = loss of heterozygosity and disease, cancer
What are the features of Spo11?
- conserved
- meiosis specific
- low sequence specifity
Why Spo11 not in mitotic cells?
DSB and dhj lead to LOH cancer/disease
What recombinase catalyses strand exchange in vitro?
RecA
What was Sehorn experiment to show strand exchange in vitro?
Used purified human-meiosis specific recombinase Dmc1
1) added Dmc1 to css and lds
2) took samples at different time points
- css and lds
- jm
- nc and lss
3) analysed products by gel electrophoresis
What challenged of dHJ?
finding correct sequences
why use homologous rather than sister chromatids
How is the correct homolgous sequence found?
recombinases allow homolgy search within 3stranded structure
Why do think that NCO products mostly generated via a hj independednt mechanism and what is this mechanism>
- most NCO products are detected before HJ formed
- synthesis dependent strand annealing
What are the key points of SDSA?
1) newly synthesised DNA displaced from template anneals to complimentary broken end
2) donor chromatid remains unchangd
What is strand return facilitated by and what proteins?
facilitated by bubble migration
helicases stop invading strand from synthesising from template DNA
What is more frequent during meiosis, gene conversions or co?
gene conversions
What are the outcomes of DSB in meiosis?
1) repaired using homolgous chromosomes
2) leading to few dHJ -> CO
3) or SDSA -> non cross overs
What are the outcomes of DSB in mitosis?
1) repair using sister chromatid
2) leads to very few dHJ -> CO and nCO
3) OR SDSA leads to nonCO
How can view CO and GC in meiotic cells?
immunoflourence analyss of human pachytene spermatocyte
antibodies stain
-synaptonemal complexes - red
-centromere - blue
-CO - green
show all bivalents have at least 1 CO nomore 3
How is the frequency of crossing over & dsb regulated?
by spo11
where Co occur, not at Spo11
where DSB form, not Spo11 nearby sites
